Quantitative Link Between Chromatin Compaction and Epigenetic Memory at the Single-cell Level

Histone modifications are often correlated to epigenetic memory: permanent changes in gene expression inherited over generations to pass information to descendants or maintain cellular identity. Repressive histone modifications such as H3K9me3 are believed to compact chromatin so that it becomes inaccessible to transcriptional machinery. However, it is not clear if compaction is needed for gene silencing or epigenetic memory. Moreover, quantitative analysis of the direct relationship between chromatin compaction and epigenetic memory is still lacking. Here, we measured 3D chromatin structure changes using multiplexed DNA FISH upon targeted epigenetic perturbations by direct recruitment of chromatin regulators to a reporter gene. We found that KRAB recruitment, known to cause H3K9me3 modifications and epigenetic memory, leads to chromatin compaction around 20kb, and that compaction is retained in permanently silenced cells even after releasing KRAB from the genome. However, recruitment of histone deacetylase HDAC4 can silence the reporter gene without resulting in chromatin compaction. More generally, by testing perturbations that cause different fractions of cells in the population to maintain epigenetic memory, we found that chromatin compaction is correlated with the amount of epigenetic memory rather than gene silencing. Finally, we found that this quantitative connection also holds in a natural context associated with irreversible transitions: epigenetic silencing and chromatin compaction at the Nanog locus in mouse ES cells during differentiation and fate commitment.